Many cardiac functions, from contractility to gene expression and structural remodeling, are regulated by multiple spatially and functionally distinct pools of cAMP. Cyclic nucleotide phosphodiesterases (PDEs), by hydrolyzing cAMP, regulate the amplitude, duration, and compartmentation of cAMP-mediated signaling. Our data suggest that PDE3A is a component of a molecular scaffold that may integrate cyclic AMP and SERCA2 transduction pathways in cardiac muscle. Immunohistochemical staining of human myocardium indicates that PDE3A is co-localized with desmin, AKAP18 and SERCA2 to sarcomere Z-bands, while PDE3B is co-localized with mitochondrial proteins (COX4, ATP synthase, Cyt-C). During sucrose gradient centrifugation of mouse cardiac membranes, PDE3A co-fractionates with sarcoplasmic reticulum (SR) Ca+2 ATPase 2 (SERCA2) and phospholamban, and immunochemical staining indicates that PDE3A co-localized with SERCA2 and desmin in mouse heart. In addition, Western blots and LC-MS/MS analysis of PDE3A immunoprecipitates indicates that murine PDE3A coimmunoprecipitated with SERCA2. Similarly, in solubilized human and murine cardiac microsomes, PDE3A co-immunoprecipitates with SERCA2 and other signaling molecules thought to be components of an AKAP/SERCA2 macromolecular regulatory complex, including phospholamban, PKARII, PP2A, and AKAP18, but not AKAP LBC. In human myocardial microsomes, the PKA catalytic subunit (PKAc) (plus ATP) phosphorylates the isoforms PDE3A1 and PDE3A2 but not PDE3A3, and significantly increases PDE3 catalytic activity. cAMP or PKAc significantly increases Ca2+ uptake into SR vesicles, and cilostamide, a PDE3-selective inhibitor, potentiates the effect of cAMP. In murine cardiac microsomes, cilostamide increased the effect of cAMP on Ca2+ uptake into SR vesicles. SERCA2 Ca2+ -ATPase activity and Ca2+ uptake and Ca2+ content were increased in SR vesicles prepared from PDE3A-knockout (KO) mice, compared to wild type. In lysates from KO hearts, SERCA2 expression was increased and that of PLB decreased, and phosphorylation of PLB at Ser-16 (pPLB/PLB ratio) was increased. In KO lysates, due to the loss of PDE3A activity, PKA was activated, as evidenced by increased phosphorylation of PKA substrates and PLB. In collaborative studies, these PDE activity changes in PDE3A-/- hearts were associated with elevations in contractility and relaxation properties of isolated hearts, as well as increased Ca2+ transient amplitudes in isolated cardiac myocytes, without differences in L-type Ca2+ currents (ICa,L). Ca2+ transients and SR Ca2+ content were normalized to the WT levels by dialysis of myocytes with the PKA inhibitor RpcAMP. PDE3 inhibition had no effect on cardiac contractility, Ca2+ transients, or SR Ca2+ content in PDE3A-/- preparations but increased these same parameters in WT hearts to levels indistinguishable from PDE3A-/-, establishing that PDE3A is the PDE3 isoform regulating basal heart function in heart, as a component of a macromolecular complex which regulates cAMP levels in microdomains containing SERCA2-PLN-PDE3A complexes. Taken together, these data suggest that, as a component of SERCA2-containing macromolecular complexes in murine and human myocardium, PDE3A regulates a discrete cAMP pool important in regulating contractility by modulating Ca2+ uptake into the SR. In addition to effects on contractility, PDE3 is thought to affect vascular smooth muscle relaxation and proliferation. The function and role of individual PDE3A and PDE3B isoforms in regulation of these processes is, however, is largely unclear, primarily due to the lack of isoform-selective PDE3 inhibitors. Using VSMCs (cultured vascular smooth muscle myocytes) expanded from the aortas of PDE3A KO and PDE3B KO mice, we examined the role of PDE3A and B isoforms in regulation of VSMC growth, and the mechanisms by which PDE3 isoforms may affect signaling pathways that mediate PDGF-induced VSMC proliferation. Cultured VSMCs expanded from the aortas of PDE3A KO mice exhibited marked inhibition of serum- and PDGF-induced DNA synthesis when compared to VSMCs expanded from PDE3A WT type and PDE3B KO mice. Growth inhibition was accompanied by selective inhibition of ERK phosphorylation in PDE3A KO VSMCs, most likely due to a combination of increased site-specific Raf-1ser259 inhibitory phosphorylation as well as excessive dephosphorylation of ERKs by elevated MKP-1 (MAP kinase phosphatase 1). Furthermore, PDE3A KO VSMCs exhibited elevated basal PKA activity, upregulation of CREB and p53 and its phosphorylation, and elevated p21 expression, together with reduction of cyclin D1 and Rb levels and Rb phosphorylation. Adenoviral infection with inactive CREB (mCREB) partially restored growth effects of serum in PDE3A KO VSMCs. In contrast, exposure of PDE3A WT VSMCs to Vp16 CREB (active CREB) was associated with inhibition of cell growth, effects similar to those observed in untreated PDE3A KO VSMCs. Transfection of PDE3A KO VSMCs with p53 siRNA reduced p21 and MKP-1 elevations and completely restored growth, without affecting cyclin D1 levels and Rb phosphorylation. We conclude that PDE3A. not PDE3B, regulates VSMC growth via two complimentary signaling pathways, i.e., PKA-mediated inhibition of MAPK signaling via Raf-1 site-specific inhibitory phosphorylation and PKA-CREB-mediated induction of p21, leading to GO/G1 cell cycle arrest, as well as by induction of p53 which induces MKP-1, p21and Wip1, leading to inhibition of G1 to S cell cycle progression. Although other studies using cAMP agonists and pharmacologic inhibitors of PDE3 and PDE4 isoforms have reported inhibition of VSMC growth, the exact role of PDE3A and PDE3B isoforms, however, was difficult to discern, in large part because of the lack of availability of isoform-specific PDE3A and PDE3B inhibitors. Taken together, our data and those of others, suggest that PDE3A isoforms may play a major role in cardiovascular function by regulating cardiac contractility and peripheral vasodilation as well as VSMC growth. Future in vivo studies with balloon injury models are needed to test the protective effect of PDE3A deletion on vessel wall thickening, smooth muscle cell migration and growth, since, under basal conditions, PDE3A KO mice do not appear to be grossly compromised in terms of growth, development and cardiovascular status. PDE3A may be an important regulator of cell cycle progression in other cells. Female PDE3A KO mice are sterile, presumably because increased cAMP/PKA signaling in oocytes maintains meiotic arrest at prophase I of the first meiotic division, and thereby inhibits oocyte maturation and prevents fertilization.Our results suggest that Polo-like kinase 1 (Plk1) may be a target of PKA, and involved in meiotic arrest of PDE3A KO oocytes. In cultured, G2/M-arrested PDE3A KO murine oocytes, elevated PKA activity was associated with inactivation of Cdc2 and Plk1, and inhibition of phosphorylation of histone H3 (S10) and of dephosphorylation of Cdc25B (S323) and Cdc2 (Thr14/Tyr15). In WT oocytes, PKA activity was transiently reduced and then increased to that observed in PDE3A KO oocytes;Cdc2 and Plk1 were activated, and phosphorylation of histone H3 (S10) and dephosphorylation of Cdc25B (S323) and Cdc2 (Thr14/Tyr15) were observed. In WT oocytes, PKAc were rapidly translocated into nucleus, and then to the spindle apparatus, but in PDE3A KO oocytes, PKAc remained in the cytosol. Plk1 was reactivated by incubation of PDE3A KO oocytes with PKA inhibitor, Rp-cAMPS. PDE3A was co-localized with Plk1 in WT oocytes, and co-immunoprecipitated with Plk1 in WT ovary and Hela cells. PKAc phosphorylated rPlk1 and Hela cell Plk1 and inhibited Plk1 activity in vitro. Taken together, our results suggest that PKA-induced inhibition of Plk1 may be critical in oocyte meiotic arrest and female infertility.